1 PROJECT SUMMARY 2 Most tuberculosis (TB) TB transmission studies to date have relied heavily on the standard culture of bacteria 3 from sputum to determine transmission links. The underlying assumption with this approach is that standard 4 culture methods allow for the recovery of all transmitted strains. Furthermore, individuals not reporting TB 5 symptoms and those with negative sputum cultures have been presumed to be non-infectious. These 6 assumptions form the fundamental premise of the contemporary study of TB transmission. Our description of 7 differentially culturable tubercle bacilli (DCTB) in sputum that are unable to grow in standard culture and only 8 emerge in liquid media supplemented with culture filtrate (CF) challenge prevailing assumptions and have 9 important implications for TB transmission. The presence of DCTB in sputum suggests that standard culture 10 fails to detect significant proportions of bacterial populations that are relevant to TB transmission. 11 We hypothesize that DCTB assays on sputum will allow for an increased yield in the quantum of bacteria 12 recovered and will identify more bacterial genotypes when compared to standard culture. This will facilitate the 13 establishment of transmission links that would be missed by standard culture. In index TB patients, we will 14 explore this, first through an in-depth characterization of bacterial genotypes that emerge in standard sputum 15 culture compared to those emerging from DCTB assays. The genotypes of the bacterial strains will be 16 identified by whole genome sequencing. We will focus both on drug sensitive and rifampin monoresistant TB, 17 the transmission of the latter identified as an emerging public health crisis in TB endemic countries. Following 18 characterization of index patients, we will use DCTB assays to increase bacterial yield and genotypic diversity 19 recovered from the sputum of household contacts. We will then use transmission inference modelling to 20 establish new transmission links in the household that were detected by DCTB assays but missed using 21 standard culture. In addition, we will test the utility of novel fluorogenic probes that label viable mycobacteria as 22 an alternate method to study very low bacterial loads in sputum of household contacts that may not be 23 detected with DCTB assays. 24 We anticipate that this approach will provide novel insight on TB transmission in the household, thereby 25 allowing for effective targeting of transmission blocking interventions to this setting. In addition, the application 26 of DCTB assays combined with in-depth whole genome sequencing of emerging isolates will validate new 27 methods to study transmission of TB.